Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
In vertebrate embryos, the neural tube displays distinct cell types at defined dorsoventral positions. Floor plate cells differentiate at the ventral midline; motor neurons appear in ventrolateral regions; and sensory relay neurons, neural crest, and roof plate cells appear dorsally. The generation of cell pattern in the neural tube depends on signals that derive from surrounding tissues. A clear example of this is the influence of axial mesoderm on the development of ventral cell types.
The differentiation of floor plate cells, motor neurons, and other ventral cell types requires inductive signals from axial mesodermal cells of the notochord. In the absence of the notochord, floor plate cells and motor neurons do not differentiate (Placzek et al., 1990b; Bovolenta and Dodd, 1991; Clarke et al., 1991; van Straaten and Hekking, 1991; Yamada et al., 1991; Ruiz i Altaba, 1992; Goulding et al., 1993; Ruiz i Altaba et al., 1993a; Halpern et al., 1993) Conversely, notochord grafts can induce the ectopic differentiation of floor plate cells and motor neurons in vivo and in vitro (van Straaten et al., 1988; Placzek et al., 1990b, 1991, 1993, Yamada et al., 1991, 1993; Ruiz l Altaba, 1992; Goulding et al., 1993). Floor plate cells themselves also possess both floor plate and motor neuron inducing activity (Yamada et al., 1991, 1993; Hatta et al., 1991; Placzek et al., 1993). In vitro assays have provided evidence that floor plate induction requires a contact-mediated signal, whereas motor neurons can be induced by diffusible signals (Yamada et al., 1993; Placzek et al., 1990b, 1993).
The differentiation of floor plate cells and motor neurons is associated with the expression of different classes of transcription factors. Floor plate cells express three members of the hepatocyte nuclear factor HNF-3/fork head gene family (Weigel and Jackie, 1990, Lai et al., 1991): Pintallavis (XFKH1/XFD1/1), HNF-3xcex2, and HNF-3xcex1 (Dirksen and Jamrich, 1992; Knochel et al., 1992; Ruiz l Altaba and Jessell, 1992; Bolce et al., 1993; Monaghan et al., 1993; Ruiz l Altaba et al., 1993a; Sasaki and Hogan, 1993; Strahle e al., 1993). Ectopic expression of Pintallavis and HNF-3xcex2 leads to the appearance of floor plate markers in cells in the dorsal region of the neural tube (Ruiz i Altaba et al., 1992, 1993b; A.R.A. et al., unpublished data; Sasaki and Hogan, 1994) suggesting that members of this family may specify floor plate cell fate. The differentiation of motor neurons is associated with expression of islet-1, a member of the LIM homeobox gene family (Ericson et a!., 1992; Yamada et al., 1993). In addition to their possible functions in cell faze determination, these transcription factors provide markers that can be used in conjunction with cell surface molecules to monitor floor plate and motor neuron differentiation.
Cell patterning in the dorsal neural tube appears to be regulated by members of two families of secreted proteins that also have prominent roles in insect development. The transforming growth factor xcex2 (TGFxcex2) family member dorsalin-1 is expressed in the dorsal neural tube and can induce the differentiation of neural crest cells in neural plate explants in vitro (Basler et al., 1993). Members of the wnt family are also expressed in the dorsal neural tube (Roelink and Nusse, 1991; Nusse and Varmus, 1992; Parr et al., 1993). In Drosophila, the TGFxcex2 family member decapentaplegic (dpp) regulates the dorsoventral pattern of the Drosophila embryo (see Ferguson and Anderson, 1992) and the differentiation and patterning of cells in imaginal discs (Spencer et al., 1982; Posakony et al., 1991; Campbell et al., 1993, Heberlein et al., 1993). similarly, wingless (wg), a member of the wnt gene family, controls cell fates during segmentation and imaginal disc development (Morata and Lawrence, 1977; Nusslein-Volhard and Wieschaus, 1980; Baker, 1988; Martinez-Arias et al., 1988; Struhl and Basler, 1993).
A third Drosophila gene important in the specification of cell identity is hedgehog (hh) (Nusslein-Volhard and Wieschaus, 1980). hh acts with dpp and wg to control cell fate and pattern during segmentation and imaginal disc development (Hidalgo and Ingham, 1990; Ingham, 1993; Ma e tal., 1993; Heberlein et al., 1993; Basler and Struhl, 1994; Heemskerk and DiNardo, 1994). hh encodes a novel protein (Lee et al., 1992; Mohler and Vani, 1992; Tabata et al., 1992; Tashiro et al., 1993) that enters the secretory pathway (Lee et al., 1992), and genetic evidence indicates the hh function is not cell autonomous (Mohler, 1988; Heberlein et al., 1993; Ma et al., 1993; Basler and Struhl, 1994), consistent with the possibility that hh acts as a signaling molecule.
The importance of hh in cell patterning in insects prompted applicants to search for vertebrate homologs and to examine their potential functions during early neural development. Applicants disclose here the cloning of a vertebrate homolog of hh, vhh-1, from rat. Recent independent studies have identified a vertebrate homolog of hh, sonic hedgehog (shh), that is closely related to vhh-1 and appears to regulate cell patterning in the neural tube and limb bud (Echelard et al., 1993; Krauss et al., 1993, Riddle et al., 1993). Here, applicants present evidence that vhh-1 is involved in the induction of ventral neural cell types. vhh-1 is expressed in midline structures (in particular, the node, notochord, and floor plate) at a time when these cells have inducing activity. COS cells expressing the rat vhh-1 gene induce floor plate and motor neuron differentiation in neural plate explants in vitro. Moreover, widespread expression of the rat vhh-1 gene in frog embryos leads to ectopic expression of the floor plate markers in the neural tube. These results suggest that vhh-1 expression in the notochord provides an inductive signal that is involved in the differentiation of floor plate cells, motor neurons, and possibly other cell types in the ventral neural tube.
This invention provides an isolated nucleic acid molecule encoding a vertebrate vhh-1 protein. In one embodiment of this invention, the nucleic acid molecule encoding a frog vhh-1 protein. In another embodiment, the nucleic acid molecule encoding a mammalian vhh-1 protein. In a further embodiment, the nucleic acid molecule encoding a rat vhh-1 protein. In a still further embodiment, the nucleic acid molecule encoding a human vhh-1 protein.
This invention provides a nucleic acid molecule comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a vertebrate vhh-1 protein.
This invention also provides monoclonal and polyclonal antibodies directed to a vhh-1 protein.
This invention provides a transgenic, nonhuman mammal comprising the isolated nucleic acid molecule encoding a vhh-1 protein.
This invention provides a method of producing a purified vertebrate vhh-1 protein which comprises: (a) inserting nucleic acid molecule encoding the vertebrate vhh-1 protein in a suitable vector; (b) introducing the resulting vector in a suitable host cell; (c) selecting the introduced host cell for the expression of the vertebrate vhh-1 protein; (d) culturing the selected cell to produce the vhh-1 protein; and (e) recovering the vhh-1 protein produced.
This invention provides a method of inducing the differentiation of floor plate cells comprising contacting floor plate cells with a purified vertebrate vhh-1 protein at a concentration effective to induce the differentiation of floor plate cells.
This invention provides a method of inducing the differentiation of floor plate cells in a subject comprising administering to the subject a purified vertebrate vhh-1 protein at an amount effective to induce the differentiation of floor plate cells in the subject.
This invention provides a method of inducing the differentiation of motor neuron comprising contacting the floor plate cells with a purified vertebrate vhh-1 protein at a concentration effective to induce the differentiation of motor neuron.
This invention provides a method of inducing the differentiation of motor neuron in a subject comprising administering to the subject a purified vertebrate vhh-1 protein at an amount effective to induce the differentiation of motor neuron in the subject.
This invention provides a method of generating ventral neurons comprising contacting progenitor cells with a purified vertebrate vhh-1 protein at a concentration effective to generate ventral neurons.
This invention provides a method of generating ventral neurons from progenitor cells in a subject comprising administering to the subject a purified vertebrate vhh-1 protein at an amount effective to generate ventral neurons from progenitor cells in the subject.
This invention provides a pharmaceutical composition comprising a vertebrate vhh-1 proein and a pharmaceutically acceptable carrier. En an embodiment, the vhh-protein is a rat protein. In another embodiment, the vhh-protein is a human protein.
This invention provides a method for generating motor neurons from undifferentiated precursor neurons consisting of introducing an amount of a pharmaceutical composition comprising the human vhh-1 protein effective to generate motor neurons from undifferentiated precursor neurons. The generation of motor neurons can alleviate acute nervous system injury or chronic neurodegenerative diseases, such as Amyotropic lateral sclerosis (ALS).
This invention provides a method of generating motor neurons from undifferentiated precursor neurons wherein the acute nervous system injury is localized to specific central axons which comprises surgical implantation of a pharmaceutical compound comprising the human vhh-1 protein and a pharmaceutically acceptable carrier effective to generate motor neurons from undifferentiated motor neurons located proximal to the injured axon(s).